Our long-term objectives are to understand the molecular virology of HCV and translate this knowledge into new antiviral strategies. Recently, the HCV protein NS4B has emerged as the key player in the induction of the membranous web, a distinct novel intracellular membrane structure seen on electron microscopy which accumulating evidence suggests represents the site of HCV replication. The molecular mechanism(s) whereby NS4B participates in this critical process is not known. We have identified two different heretofore unrecognized structural motifs within NS4B that are essential for mediating NS4B's role in HCV replication: a N-terminal amphipathic helix (AH) that is sufficient for mediating membrane association and a nucleotide binding motif (NBM) that is shared with other proteins known to bind and hydrolyze nucleotides such as GTP and ATP. Both the AH and NBM are conserved across natural HCV isolates. Mutation of specific amino acids within each of these motifs results in dramatic inhibition of HCV RNA replication. We hypothesize: 1) the minimal elements required for membrane association or RNA replication can be further narrowed to a subset of amino acids within the AH; 2) the nucleotide-binding motif within NS4B reflects a biochemical activity that is required for RNA replication; 3) one or both of these domains mediates formation of the membranous web; 4) selected mutations in these domains may have transdominant effects on HCV replication; 5) the AH-mediated membrane association of NS4B and the NBM can be pharmacologically disrupted. We will test these hypotheses by using mutagenesis, electron and immuno-microscopy, and biochemical assays for membrane association and interaction with nucleotides to further study the mechanism of NS4B's membrane association and NBM activity. The consequences of disrupting these domains on the formation of membranous web replication sites will also be examined. HCV replicons will be used to identify NS4B's cis and trans functions in RNA replication. High-affinity inhibitory ligands of AH and NBM function will be developed using phage display. Finally, we will explore how these findings can be translated into novel anti-HCV strategies.